Bacterial acquisition of resistance determinants represents a major threat to human health. The recent discovery of cfr (chloramphenicol-florfenicol resistance gene) in a multidrug-resistant hospital isolate of Staphylococcus aureus is an important recent example of bacterial resistance. Furthermore, the presence of this gene on mobile genetic elements raises the possibility of the spread of the resistance among human pathogens, an implication that could have devastating effects on human health. The bacterial resistance mediated by cfr is a consequence of an unprecedented target modification strategy. Cfr encodes a methyltransferase enzyme which catalyses addition of a methyl group to adenosine 2503 (A2503) in ribosomal RNA. This nucleotide is positioned in the peptidyl transferase center of the large ribosomal subunit, a common antibiotic target, and its modification by Cfr precludes binding of antibiotics. Cfr and its evolutionary relative RlmN are members of the Radical SAM (S-adenosyl methionine) superfamily. Both enzymes modify amidine carbons in the substrate adenosine: while RlmN transfers the methyl group to the C2 position, Cfr methylates C8 carbon. The formation of a carbon-carbon bond between the aromatic amidine carbon and the methyl group is an unprecedented bond-forming event in enzymology. The goal of this application is to define the mechanism of this novel mode of methylation. The central hypothesis is that methylation is enabled by the enzyme's ability to use two molecules of SAM per each methyl group introduced: one as a cofactor and a source of the reactive 5'-deoxyadenosyl radical, and the other as a cosubstrate and a source of newly added carbon, a hypothesis formulated on the basis of our preliminary data. The following specific aims will be investigated: 1. mechanistically informative substrate analogues will be used to define the chemical mechanism of the reaction; 2. Roles of catalytically crucial conserved residues in methyltransferases will be interrogated by site-directed mutagenesis; and 3. the specificity of both enzymes towards A2503 will be investigated through substrate modulation. The approach is innovative because it addresses a novel and unique mode of enzymatic catalysis, as predicted by the preliminary data. The proposed research is significant because it adds an unprecedented function to the Radical SAM superfamily. Moreover, the proposed research is expected to advance and expand understanding of modes of enzymatic methylation in biology. Ultimately, detailed understanding of this mechanism has the potential to inform the development of next generation antibiotics that will help alleviate the growing problem of antibiotic resistance.